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Microbial deterioration of traditional alcoholic beverages in Nigeria
Food Research International 32 (1999) 163±167
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Microbial deterioration of traditional alcoholic beverages in Nigeria A.I. Sanni*, A.A. Onilude, I.F. Fadahunsi, R.O. Afolabi Food and Applied Microbiology Research Group, Department of Botany and Microbiology, University of Ibadan, Nigeria Received 30 April 1998; accepted 1 May 1999
Abstract A study was carried out to investigate the microbiological quality and physico-chemical changes of ``aged'' cereal-based traditional alcoholic beverages in Nigeria. There were variations in the microorganisms isolated from sekete, pito and burukutu at postproduction time of 72 h, but Saccharomyces cerevisiae, Acetobacter aceti, A. hansenii, A. pasteurianus, Alcaligenes, Flavobacterium, Lactobacillus plantarum and L. brevis were common to all samples. A vinegary ¯avour and o-odour were the pronounced characteristics of the deteriorating beverages. The decrease in the ethanol from 3% in the fresh product to 1% at the end of 72 h retailing period is proportional to the acetic acid content of the drinks. However, slight decreases were observed for lactic, malic, succinic and formic acid constituents of the beverages. Although no enterobacteriaceae was isolated, some of the identi®ed isolates are potentially pathogenic, and can become injurious to the health of the consumers of ``aged'' batches of the beverages. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved.
1. Introduction Various kinds of traditional alcoholic beverages are produced in Nigeria and other West African countries from many types of agricultural sources. The beverages can be conveniently categorised as follows. (i) Those produced from fermented sugary sap or fruit juice; this includes palm wine from palm tree (Elaeis guineensis) or raa palm (Raphia hookeri) and agadagidi from ripe plantain pulp. The drinks are milky white, eervescent with sweet taste (Faparusi, 1971; Okafor, 1972; Sanni & Oso, 1988). (ii) Those produced from malted cereal grains; these are pito (Campbell-Platt, 1987; Demuyakor & Ohta 1991; Ekundayo, 1969; Hulse et al., 1980; Sefa-Dedeh & Asante, 1988) burukutu (Faparusi, Olo®nboba & Ekundayo, 1973; Sefa-Dedeh & Asante, 1988) akadamu and sekete (Sanni, 1989; Sefa-Dedeh & Mensah, 1989). In Ghana, there are four types of pito Ð Nandom, Kokomba, Togo and Dagarti. The peculiar characteristics of each lies in their wort extraction and inoculum sources.The cereal-based beverages have heavy consistency because of suspended undigested starch granules, and the presence of microorganisms responsible for the fermentations (Okafor, 1990). * Corresponding author. Tel.: +234-2-810110-4, ext. 1403; fax: +234-2-8103043/8103118. E-mail address: [email protected] (A.I. Sanni)
The production of these beverages like any other traditional fermented foods in Africa is by the age-long method of chance inoculation and uncontrolled fermentation process. Therefore, the usual variations in the quality and stability of the products are not unexpected. Studies over the years have been devoted to the isolation and identi®cation of microorganisms responsible for the various fermentations. Nutritional status of some of the products are also documented in the previous works cited. The volume of each beverage produced will depend on a number of factors. For example, the volume of palm wine available to a retailer per trip will depend on the number of palm tapped, the amount of sap that trickled into the collecting vessel and the ease of transportation to the retailing outlet. For the cereal-based beverages, a batch is usually produced from 10 to 25 kg of the grains with a yield of 4.4±5.3 l of product per kg of the substrate (Sefa-Dedeh & Asante, 1988). Although, there is no available information on the production volume of the beverages, the consuming population is large. The beverages have short shelf-life and undergo rapid deterioration within 48 h of production. This results into heavy losses being incurred by the local brewers since the unsold batches have to be discarded. Although the drinks would not normally attract consumers after 24 h of production, there are cases of special requests for batches that have ``aged'' for 48 h or more because
0963-9969/99/$20.00 # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. PII: S0963-9969(99)00068-X
164
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
of the ``harsh'' taste and assumed increase in the alcoholic content. Since these beverages are products of spontaneous fermentations, the origin and types of associated micro-organisms are diverse. Therefore, establishment of undesirable microorganisms that may be potentially pathogenic cannot be ruled out in some of the ``aged'' batches through microbial succession. Some of these microorganisms may be capable of producing toxins that could be injurious to the health of the consumers since the beverages are normally consumed together with the microorganisms responsible for the fermentation. In this paper, information is provided on the microorganisms associated with three deteriorating Nigerian traditional beverages produced from malted cereal grains. The observed physico-chemical changes in the products are also reported. 2. Materials and methods 2.1. Traditional preparation of the beverages Generally, the production process for the three beverages involved washing of the cereal grains, steeping in water for about 2 days followed by malting which is carried out on moistened banana leaves or jute bags for 3 to 4 days with intermittent watering. Drying and milling of the germinated grains are carried out and the wort extracted by boiling. The wort is allowed to cool, poured into earthenware pots and left to ferment for 2±3 days. A portion of the previous brew is usually added to initiate fermentation. The alcohol content of the products may vary from 2 to 3%.
and Plate Count Agar for viable counts. All incubations were carried out at 30 C for 48 h. 2.4. Characterisation and identi®cation of microorganisms Preliminary morphological and microscopic examinations were done on the bacteria isolates. The organisms were stained by Gram's method and tested for catalase reaction and endospore forming ability. Gram's positive and catalase negative rods and cocci were categorised on the basis of their carbohydrate fermentation ability using API 50 CH and 50 CHL kits (API System, Montalieu, Vercieu, France). The data were subjected to numerical analysis using clustan 2 computer software (Release 1.2, St. Andrews Fife, UK). The catalase positive, endospore-formers were identi®ed by the method of Gordon, Haynes and Pang, (1973), while acetic acid bacteria isolated on Frauters medium were characterised using the methods of Carr (1968) and De Ley, Gillis and Swings, (1986). Other bacteria isolates were identi®ed using conventional methods and con®rmation of identi®cation was by reference to standard descriptions. Identi®cation of yeast isolates was based on the modi®ed scheme of Deak and Beuchat (1987), but carbon assimilation test was on API ATB 32 C. Con®rmation of identi®cation was by comparing the observed characteristics with the standard descriptions in Kregervan Rij (1984). 2.5. Determination of pH and viable counts
Samples of burukutu, sekete and pito were collected from ®ve local producers located in three geographical areas in south-western Nigeria. A total of three samples of each beverage were collected at the end of the 72 h fermentation period (fresh samples) and at the 72 h of post-production (deteriorating samples).
The pH of the fresh and deteriorating beverage samples was determined using the combined glass±calomel electrode and a pH meter PHM 61 (Radiometer, Copenhagen, Denmark). For the viable counts, samples were treated in ultrasonic bath for 5 min before mixing thoroughly on Vortex for 2 min. Colony Forming Units (CFU/g) were obtained on Plate Count Agar (total count), MRS agar at pH 5.5 (lactic acid bacteria), Frauters medium (acetic acid bacteria) and MEA supplemented with streptomycin sulphate (yeasts). Incubation was at 30 C for 48 h.
2.3. Isolation procedure
2.6. Chemical analysis
Isolation of micro-organisms from the fresh and deteriorating beverage samples were carried out immediately on the following media: Nutrient Agar for bacteria, MRS agar at pH 5.5; (supplemented with cycloheximide) for lactic acid bacteria, Malt Extract Agar (supplemented with streptomycin sulphate) for yeasts, Frauter's ethanol medium (Frauters, 1950) containing yeast extract, 10 g; CaCO3, 20 g; ethanol, 20 g; agar, 20 g in 1 l of distilled water for isolation of acetic acid bacteria, VRBG for the isolation of enterobacteriaceae
Duplicate samples of the fresh and deteriorating beverages were prepared for analysis according to the method of Lonner and Preve-Akesson (1988). A 10 g amount of each sample was mixed with 90 ml distilled water and homogenised by the use of a stomacher (Lab Blender model 80. Seward Medical, London) for 30 s `normal' speed. The mixture was heated to 60 C for 5 min and cooled to room temperature in an ice bath. The pH was adjusted to 7 with 0.1 N NaOH and stirred for 30 min. Samples were centrifuged for 5 min at 30 C
2.2. Sample collection
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
165
Table 1 Microorganisms isolated from the deteriorating beverage samples Product
Organisms
Substrate
Nature of product
Sekete
A. aceti, A. pasteurianus, L. brevis, L. buchneri, L. plantarum, Lactobacillius spp., S. cerevisiae, Saccharomyces spp., Flavobacterium spp., Micrococcus varians, B. licheniformis
Maize
Light brown liquid
Pito
A. aceti, A. hansenii, A. pasteurianus, L. plantarum, L. fermentum, L. brevis, Alcaligenes, S. cerevisiae, Micrococcus spp., Candida spp., B. licheniformis, Flavobacterium spp.
Maize or sorghum
Dark brown liquid
Burukutu
A. hansenii, A. pasteurianus, A. aceti, Candida mycoderma, Hansenula anomala, L. brevis, L. plantarum, L. fermentum, Alcaligenes, S. diastaticus (questionable), Bacillus spp., Rhodoto spp., rula spp.
Sorghum and gari, a fermented cassava product as adjunct
Creamy liquid
10,000 g and ®ltered through 0.2 mm membrane ®lter (Schleicher and Schuell, Dassel, Germany) and stored at 20 C. Analysis was on Shimadzu HPLC-6A equipped with a hydrogen loaded ion exchange column, Bio-Rad Aminex X-87H Bio-Rad, Richmond, CA) and a Shimadzu Rid 6A refractive index detector. Triplicate determinations were carried out, and a paired t-test at P0.01) was conducted on the data obtained. 3. Results and discussion The most discernible characteristics of the deteriorating pito, burukutu and sekete were vinegary ¯avour, discoloration and o-odour when compared with the fresh samples. The associated micro-organisms vary but there was common occurrence of acetic acid bacteria in all the samples (Table 1). With the exception of the Acetobacter spp. (Table 3), the biochemical properties of other micro-organisms are not presented. From previous reports (Ekundayo, 1969; Faparusi et al., 1973; Okafor, 1972; Sanni, 1989), some of the isolates have been identi®ed in the fresh products but Acetobacter spp. seem to have become established 3±4 days after the production of the beverages. Lactobacillus brevis was consistently isolated and together with the acetic acid bacteria, dominated the micro-¯ora of the deteriorating samples. Saccharomyces cerevisiae which has been reported to be responsible for the signi®cant amount of ethanol of beverages occurred consistently in the deteriorating samples. A S. diastaticus-like organism was isolated from burukutu but the identi®cation was doubtful because the observed characteristics did not agree with the standard description. The isolates' response on soluble starch was inconsistent, therefore, it is likely to be a variant of S. cerevisiae. L. brevis, L. fermentum and L. buchneri (Table 1) are known to be potential beer spoilers. They are known to cause turbidity accompanied by o-¯avour and acidity in beer.
Moulds were not within detectable limit (<102) in the samples due to the low oxygen tension of the fermenting medium. The vinegary ¯avour of the spoiled samples which was the most obvious characteristics was brought about by the acetic acid bacteria. They are usually found as contaminants of various fermented liquors (Carr, 1968). Although Acetobacter spp., L. pastorianus and Bacillus licheniformis are known to cause ropiness (Shimwell, 1947), the beverages examined in this study did not display ``lumpy'' characteristics of beer made ropy by these organisms. The ``Newtonian'' viscosity of the deteriorating beverages was not greater than that of the fresh products. There was no isolation of any enterobacteriaceae from the samples. The acidic pH of the beverages could have been inhibitory to the establishment of these Table 2 Viable counts of microorganisms in the fresh and deteriorating beverage samplesa Colony forming units (CFU/g) Beverage
TC
LAB
AAB
YEA
Sekete FS DS t 0:01
4.2109b 3.9109 (s)c
3.6105 3.4103 (s)
1.0102 1.4104 (s)
4.3105 3.2102 (s)
Pito FS DS t 0:01
4.3109 3.6109 (s)
3.4105 3.2103 (s)
1.2102 2.8104 (s)
4.1103 3.2102 (s)
Burukutu FS DS t 0:01
4.7109 4.1109 (s)
3.1105 3.1103 (s)
0.9102 3.1104 (s)
4.0102 3.4102 (s)
a
TC=total count; LAB=lactic acid bacteria; AAB=acetic acid bacteria; YEA=yeasts; FS=fresh samples; DS=deteriorating sample. b Means of triplicate determinations. c s=Signi®cant dierence between FS and DS.
166
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
Table 3 Physico-chemical changes in the fresh and deteriorating beverage samplesa Organic acids (g/100 g) Beverage pH
Ethanol (%) Lactic Acetic Formic Malic Succinic
Sekete FS DS t 0:01
3.4b 3.0 3.2 1.1 (s)c (s)
0.44 0.36 (s)
0.07 0.24 (s)
n.d. n.d. ±
0.10 0.06 (s)
0.20 0.17 (s)
Pito FS DS t 0:01
3.6 2.8 3.1 1.2 (ns)d (ns)
0.31 0.26 (ns)
0.08 0.20 (s)
n.d. n.d. ±
0.11 0.06 (s)
0.13 0.08 (ns)
0.35 0.28 (s)
0.10 0.32 (s)
0.20 0.04 (s)
0.14 0.08 (s)
0.18 0.10 (s)
Burukutu FS 3.6 DS 3.2 t 0:01 (s) a b c d
3.1 1.1 (s)
n.d.=not detected; FS=fresh sample; DS=deteriorating sample. Means of triplicate determinations. s=signi®cant dierence between FS and DS (P40.01). ns=no signi®cant dierence between FS and DS (P>0.01).
groups of microorganisms. However, some of the isolates identi®ed, e.g. Flavobacterium and Alcaligenes are potentially pathogenic and could cause gastro-intestinal disorder. There was signi®cant dierence in the viable counts of microorganisms in the deteriorating beverage samples (Table 2). The reduction in the microbial population obtained for the deteriorating beverages indicated the gradual dying o of some initial micro-¯ora of the fermentation. There were signi®cant dierences in the lactic, malic, succinic and acetic acids content of the fresh and deteriorating sekete and burukutu. However, with the exception of malic and acetic, the results obtained for the pH, ethanol, lactic acid and succinic acid for pito samples were not signi®cantly dierent (Table 3). Decrease in the ethanol content of the deteriorating beverages is attributable to the activities of the Acetobacter spp. which oxidises the ethanol to acetic acid (Table 4). The proportional increase of the acetic acid of the deteriorating beer samples led credence to the above. It should be mentioned here that strict anaerobic
Table 4 Biochemical properties of Acetobacter species isolated from the deteriorating beverages (n=9)a Isolates Tests
A
B
C
D
E
F
G
H
I
J
Gram's reaction
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
Cell dimension width (m) length (m) Morphology Catalase Nitrate reduction Acid from glucose Cellulose production Brown pigment Ethanol oxidation Growth in Hoyers medium
0.52 1.07 R + ÿ + ÿ ÿ + +
0.63 0.94 R + ÿ + ÿ ÿ + w+
0.71 1.23 R + ÿ + ÿ ÿ + +
0.56 0.86 O + ÿ ÿ ÿ ÿ + +
0.52 1.34 R + ÿ ÿ ÿ ÿ + +
0.71 1.21 R + ÿ + ÿ ÿ + +
0.62 0.94 R + ÿ + ÿ ÿ + ÿ
0.58 1.12 O + ÿ + ÿ ÿ + ÿ
0.52 0.81 R + ÿ + ÿ ÿ + ÿ
0.61 1.31 O + w+ ÿ ÿ ÿ + +
Ketogenesis from: Glycerol Mannitol Sorbitol
+ + +
+ ÿ +
+ + +
+ ÿ +
+ + +
+ + +
+ ÿ ÿ
ÿ + +
ÿ ÿ ÿ
ÿ ÿ ÿ
Ferric chloride reaction on: Glucose Fructose Galactose
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
Growth on carbon sources: Ethanol Xylose Arabinose Glucose Ranose Sodium acetate Sodium citrate
+ ÿ + + ÿ + ÿ
+ + ÿ + ÿ + ÿ
+ ÿ ÿ + ÿ + ÿ
+ ÿ ÿ ÿ ÿ + ÿ
+ ÿ ÿ + ÿ + ÿ
+ ÿ ÿ + ÿ ÿ ÿ
+ ÿ ÿ + ÿ ÿ ÿ
+ ÿ ÿ + ÿ ÿ ÿ
+ + ÿ ÿ ÿ + ÿ
+ ÿ ÿ ÿ ÿ + ÿ
Growth on 0.001% malachite green Resistance to streptomycin
ÿ +
ÿ +
ÿ +
ÿ +
+ +
+ +
ÿ +
+ +
+ +
+ ÿ
Probability identity
AC
AC
AC
AC
AC
AH
AH
AH
AP
AP
a
R=rod; O=ovoid; ÿ=negative; +=positive; w+=weakly positive; AC=Acetobacter aceti; AH=Acetobacter hansenii; AP=Acetobacter pasteurianus.
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
conditions are never achieved during the traditional brewing of these beverages, thus creating enabling environment for the acetic acid bacteria to become established and initiate spoilage. Despite the problem of spoilage, the production of these beverages oer an avenue for employment and income for the producers. According to Sefa-Dedeh and Asante (1988), production of pito in Ghana not only provides psychological and social enjoyment, but has important implications for the food system and economy of the country. Acknowledgements A.I.S. acknowledges the award of a Guest Scholarship by the Swedish Institute for research studies in the Department of Applied Microbiology, University of Lund, Sweden, where some aspects of this work were concluded. References Campbell-Platt, G. (1987). Fermented foods of the world, a dictionary guide. London: Butterworth, (pp. 193±195). Carr, J. G. (1968). Methods for identifying acetic acid bacteria. In B. M. Gibbs, & D.A Shapton, Identi®cation methods for microbiologists Part B. (p. 212). London, New York: Academic Press. Deak, T., & Beuchat, R. (1987). Identi®cation of food borne yeasts. Journal of Food Protection, 50, 243±264. Demuyakor, B, & Ohta, Y. (1991). Characteristics of pito yeast's from Ghana. Food Microbiology, 8, 183±193. Dey Ley, J., Gillis, M., & Swings, J. (1986). Family VI acetobacteraceae. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, & J. G. Holts, Bergey's manual of systematic bacteriology (pp. 267±278). Baltimore: Williams and Wilkins.
167
Ekundayo, J. A. (1969). The production of pito, a Nigerian fermented beverage. Journal of Food Technology, 4, 217±225. Faparusi, S. I. (1971). Micro¯ora of fermenting palm sap. Jour. Fd. Sci. Technol, 8, 206. Faparusi, S. I., Olo®nboba, M. O., & Ekundayo, J. A. (1973). The microbiology of burukutu beer. Zeitschrift fur Allgemaine Mikrobiologie, 13, 563±568. Frauters, J. (1986). Family VI acetobacteraceae.. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, & J. G. Holts, Bergey's Manual of Systematic Bacteriology (pp. 267±278). Baltimore: Williams and Wilkins. Gordon, R.E., Haynes, W.C., & Pang, C.H. (1973). The genus Bacillus. In USDA agricultural handbook, No. 427. Washington, DC. Hulse, J. H., Laing, E. M., & Pearson, O. E. (1980). Sorghum and the millets: their composition and nutritive value. London: Academic Press. Kreger-van Rij, N. J. W. (1984). The yeasts, a taxonomic study. Amsterdam: Elsevier Science. Lonner, C., & Preve-Akesson, K. (1988). Acidi®cation properties of lactic acid bacteria in rye sour dough's. Food Microbiology, 5, 43± 58. Okafor, N. (1972). Palm wine yeast's from parts of Nigeria. Journal of Science of Food and Agriculture, 23, 1399±1402. Okafor, N. (1990). Traditional alcoholic beverages of Tropical Africa: strategies for scale up. Process Biochemistry International, December 213±220. Sanni, A. I. (1989). Chemical studies on sekete beer. Food Chemistry, 33, 187±191. Sanni, A. I., & Oso, B. A. (1988). The production of agadagidi, a Nigerian fermented beverage. Die Nahrung, 32, 319±326. Sefa-Dedeh, S., & Asante, S. (1988). Traditional alcoholic beverages from cereals in Ghana: processing of pito and burukutu in the Greater Accra Region, Ghana. KIFP Food Systems Research Report, No 1. Sefa-Dedeh, S., & Mensah, E. (1989). Cereal processing methods in Greater Accra Region, Ghana. KIFP in Food Systems Research Report, No 4. Shimwell, J. L. (1947). A study of ropiness in beer: the predisposition of beer to ropiness. Jour. Inst. Brewing, 44, 280.
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Microbial deterioration of traditional alcoholic beverages in Nigeria A.I. Sanni*, A.A. Onilude, I.F. Fadahunsi, R.O. Afolabi Food and Applied Microbiology Research Group, Department of Botany and Microbiology, University of Ibadan, Nigeria Received 30 April 1998; accepted 1 May 1999
Abstract A study was carried out to investigate the microbiological quality and physico-chemical changes of ``aged'' cereal-based traditional alcoholic beverages in Nigeria. There were variations in the microorganisms isolated from sekete, pito and burukutu at postproduction time of 72 h, but Saccharomyces cerevisiae, Acetobacter aceti, A. hansenii, A. pasteurianus, Alcaligenes, Flavobacterium, Lactobacillus plantarum and L. brevis were common to all samples. A vinegary ¯avour and o-odour were the pronounced characteristics of the deteriorating beverages. The decrease in the ethanol from 3% in the fresh product to 1% at the end of 72 h retailing period is proportional to the acetic acid content of the drinks. However, slight decreases were observed for lactic, malic, succinic and formic acid constituents of the beverages. Although no enterobacteriaceae was isolated, some of the identi®ed isolates are potentially pathogenic, and can become injurious to the health of the consumers of ``aged'' batches of the beverages. # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved.
1. Introduction Various kinds of traditional alcoholic beverages are produced in Nigeria and other West African countries from many types of agricultural sources. The beverages can be conveniently categorised as follows. (i) Those produced from fermented sugary sap or fruit juice; this includes palm wine from palm tree (Elaeis guineensis) or raa palm (Raphia hookeri) and agadagidi from ripe plantain pulp. The drinks are milky white, eervescent with sweet taste (Faparusi, 1971; Okafor, 1972; Sanni & Oso, 1988). (ii) Those produced from malted cereal grains; these are pito (Campbell-Platt, 1987; Demuyakor & Ohta 1991; Ekundayo, 1969; Hulse et al., 1980; Sefa-Dedeh & Asante, 1988) burukutu (Faparusi, Olo®nboba & Ekundayo, 1973; Sefa-Dedeh & Asante, 1988) akadamu and sekete (Sanni, 1989; Sefa-Dedeh & Mensah, 1989). In Ghana, there are four types of pito Ð Nandom, Kokomba, Togo and Dagarti. The peculiar characteristics of each lies in their wort extraction and inoculum sources.The cereal-based beverages have heavy consistency because of suspended undigested starch granules, and the presence of microorganisms responsible for the fermentations (Okafor, 1990). * Corresponding author. Tel.: +234-2-810110-4, ext. 1403; fax: +234-2-8103043/8103118. E-mail address: [email protected] (A.I. Sanni)
The production of these beverages like any other traditional fermented foods in Africa is by the age-long method of chance inoculation and uncontrolled fermentation process. Therefore, the usual variations in the quality and stability of the products are not unexpected. Studies over the years have been devoted to the isolation and identi®cation of microorganisms responsible for the various fermentations. Nutritional status of some of the products are also documented in the previous works cited. The volume of each beverage produced will depend on a number of factors. For example, the volume of palm wine available to a retailer per trip will depend on the number of palm tapped, the amount of sap that trickled into the collecting vessel and the ease of transportation to the retailing outlet. For the cereal-based beverages, a batch is usually produced from 10 to 25 kg of the grains with a yield of 4.4±5.3 l of product per kg of the substrate (Sefa-Dedeh & Asante, 1988). Although, there is no available information on the production volume of the beverages, the consuming population is large. The beverages have short shelf-life and undergo rapid deterioration within 48 h of production. This results into heavy losses being incurred by the local brewers since the unsold batches have to be discarded. Although the drinks would not normally attract consumers after 24 h of production, there are cases of special requests for batches that have ``aged'' for 48 h or more because
0963-9969/99/$20.00 # 1999 Published by Elsevier Science Ltd on behalf of the Canadian Institute of Food Science and Technology. All rights reserved. PII: S0963-9969(99)00068-X
164
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
of the ``harsh'' taste and assumed increase in the alcoholic content. Since these beverages are products of spontaneous fermentations, the origin and types of associated micro-organisms are diverse. Therefore, establishment of undesirable microorganisms that may be potentially pathogenic cannot be ruled out in some of the ``aged'' batches through microbial succession. Some of these microorganisms may be capable of producing toxins that could be injurious to the health of the consumers since the beverages are normally consumed together with the microorganisms responsible for the fermentation. In this paper, information is provided on the microorganisms associated with three deteriorating Nigerian traditional beverages produced from malted cereal grains. The observed physico-chemical changes in the products are also reported. 2. Materials and methods 2.1. Traditional preparation of the beverages Generally, the production process for the three beverages involved washing of the cereal grains, steeping in water for about 2 days followed by malting which is carried out on moistened banana leaves or jute bags for 3 to 4 days with intermittent watering. Drying and milling of the germinated grains are carried out and the wort extracted by boiling. The wort is allowed to cool, poured into earthenware pots and left to ferment for 2±3 days. A portion of the previous brew is usually added to initiate fermentation. The alcohol content of the products may vary from 2 to 3%.
and Plate Count Agar for viable counts. All incubations were carried out at 30 C for 48 h. 2.4. Characterisation and identi®cation of microorganisms Preliminary morphological and microscopic examinations were done on the bacteria isolates. The organisms were stained by Gram's method and tested for catalase reaction and endospore forming ability. Gram's positive and catalase negative rods and cocci were categorised on the basis of their carbohydrate fermentation ability using API 50 CH and 50 CHL kits (API System, Montalieu, Vercieu, France). The data were subjected to numerical analysis using clustan 2 computer software (Release 1.2, St. Andrews Fife, UK). The catalase positive, endospore-formers were identi®ed by the method of Gordon, Haynes and Pang, (1973), while acetic acid bacteria isolated on Frauters medium were characterised using the methods of Carr (1968) and De Ley, Gillis and Swings, (1986). Other bacteria isolates were identi®ed using conventional methods and con®rmation of identi®cation was by reference to standard descriptions. Identi®cation of yeast isolates was based on the modi®ed scheme of Deak and Beuchat (1987), but carbon assimilation test was on API ATB 32 C. Con®rmation of identi®cation was by comparing the observed characteristics with the standard descriptions in Kregervan Rij (1984). 2.5. Determination of pH and viable counts
Samples of burukutu, sekete and pito were collected from ®ve local producers located in three geographical areas in south-western Nigeria. A total of three samples of each beverage were collected at the end of the 72 h fermentation period (fresh samples) and at the 72 h of post-production (deteriorating samples).
The pH of the fresh and deteriorating beverage samples was determined using the combined glass±calomel electrode and a pH meter PHM 61 (Radiometer, Copenhagen, Denmark). For the viable counts, samples were treated in ultrasonic bath for 5 min before mixing thoroughly on Vortex for 2 min. Colony Forming Units (CFU/g) were obtained on Plate Count Agar (total count), MRS agar at pH 5.5 (lactic acid bacteria), Frauters medium (acetic acid bacteria) and MEA supplemented with streptomycin sulphate (yeasts). Incubation was at 30 C for 48 h.
2.3. Isolation procedure
2.6. Chemical analysis
Isolation of micro-organisms from the fresh and deteriorating beverage samples were carried out immediately on the following media: Nutrient Agar for bacteria, MRS agar at pH 5.5; (supplemented with cycloheximide) for lactic acid bacteria, Malt Extract Agar (supplemented with streptomycin sulphate) for yeasts, Frauter's ethanol medium (Frauters, 1950) containing yeast extract, 10 g; CaCO3, 20 g; ethanol, 20 g; agar, 20 g in 1 l of distilled water for isolation of acetic acid bacteria, VRBG for the isolation of enterobacteriaceae
Duplicate samples of the fresh and deteriorating beverages were prepared for analysis according to the method of Lonner and Preve-Akesson (1988). A 10 g amount of each sample was mixed with 90 ml distilled water and homogenised by the use of a stomacher (Lab Blender model 80. Seward Medical, London) for 30 s `normal' speed. The mixture was heated to 60 C for 5 min and cooled to room temperature in an ice bath. The pH was adjusted to 7 with 0.1 N NaOH and stirred for 30 min. Samples were centrifuged for 5 min at 30 C
2.2. Sample collection
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
165
Table 1 Microorganisms isolated from the deteriorating beverage samples Product
Organisms
Substrate
Nature of product
Sekete
A. aceti, A. pasteurianus, L. brevis, L. buchneri, L. plantarum, Lactobacillius spp., S. cerevisiae, Saccharomyces spp., Flavobacterium spp., Micrococcus varians, B. licheniformis
Maize
Light brown liquid
Pito
A. aceti, A. hansenii, A. pasteurianus, L. plantarum, L. fermentum, L. brevis, Alcaligenes, S. cerevisiae, Micrococcus spp., Candida spp., B. licheniformis, Flavobacterium spp.
Maize or sorghum
Dark brown liquid
Burukutu
A. hansenii, A. pasteurianus, A. aceti, Candida mycoderma, Hansenula anomala, L. brevis, L. plantarum, L. fermentum, Alcaligenes, S. diastaticus (questionable), Bacillus spp., Rhodoto spp., rula spp.
Sorghum and gari, a fermented cassava product as adjunct
Creamy liquid
10,000 g and ®ltered through 0.2 mm membrane ®lter (Schleicher and Schuell, Dassel, Germany) and stored at 20 C. Analysis was on Shimadzu HPLC-6A equipped with a hydrogen loaded ion exchange column, Bio-Rad Aminex X-87H Bio-Rad, Richmond, CA) and a Shimadzu Rid 6A refractive index detector. Triplicate determinations were carried out, and a paired t-test at P0.01) was conducted on the data obtained. 3. Results and discussion The most discernible characteristics of the deteriorating pito, burukutu and sekete were vinegary ¯avour, discoloration and o-odour when compared with the fresh samples. The associated micro-organisms vary but there was common occurrence of acetic acid bacteria in all the samples (Table 1). With the exception of the Acetobacter spp. (Table 3), the biochemical properties of other micro-organisms are not presented. From previous reports (Ekundayo, 1969; Faparusi et al., 1973; Okafor, 1972; Sanni, 1989), some of the isolates have been identi®ed in the fresh products but Acetobacter spp. seem to have become established 3±4 days after the production of the beverages. Lactobacillus brevis was consistently isolated and together with the acetic acid bacteria, dominated the micro-¯ora of the deteriorating samples. Saccharomyces cerevisiae which has been reported to be responsible for the signi®cant amount of ethanol of beverages occurred consistently in the deteriorating samples. A S. diastaticus-like organism was isolated from burukutu but the identi®cation was doubtful because the observed characteristics did not agree with the standard description. The isolates' response on soluble starch was inconsistent, therefore, it is likely to be a variant of S. cerevisiae. L. brevis, L. fermentum and L. buchneri (Table 1) are known to be potential beer spoilers. They are known to cause turbidity accompanied by o-¯avour and acidity in beer.
Moulds were not within detectable limit (<102) in the samples due to the low oxygen tension of the fermenting medium. The vinegary ¯avour of the spoiled samples which was the most obvious characteristics was brought about by the acetic acid bacteria. They are usually found as contaminants of various fermented liquors (Carr, 1968). Although Acetobacter spp., L. pastorianus and Bacillus licheniformis are known to cause ropiness (Shimwell, 1947), the beverages examined in this study did not display ``lumpy'' characteristics of beer made ropy by these organisms. The ``Newtonian'' viscosity of the deteriorating beverages was not greater than that of the fresh products. There was no isolation of any enterobacteriaceae from the samples. The acidic pH of the beverages could have been inhibitory to the establishment of these Table 2 Viable counts of microorganisms in the fresh and deteriorating beverage samplesa Colony forming units (CFU/g) Beverage
TC
LAB
AAB
YEA
Sekete FS DS t 0:01
4.2109b 3.9109 (s)c
3.6105 3.4103 (s)
1.0102 1.4104 (s)
4.3105 3.2102 (s)
Pito FS DS t 0:01
4.3109 3.6109 (s)
3.4105 3.2103 (s)
1.2102 2.8104 (s)
4.1103 3.2102 (s)
Burukutu FS DS t 0:01
4.7109 4.1109 (s)
3.1105 3.1103 (s)
0.9102 3.1104 (s)
4.0102 3.4102 (s)
a
TC=total count; LAB=lactic acid bacteria; AAB=acetic acid bacteria; YEA=yeasts; FS=fresh samples; DS=deteriorating sample. b Means of triplicate determinations. c s=Signi®cant dierence between FS and DS.
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A.I. Sanni et al. / Food Research International 32 (1999) 163±167
Table 3 Physico-chemical changes in the fresh and deteriorating beverage samplesa Organic acids (g/100 g) Beverage pH
Ethanol (%) Lactic Acetic Formic Malic Succinic
Sekete FS DS t 0:01
3.4b 3.0 3.2 1.1 (s)c (s)
0.44 0.36 (s)
0.07 0.24 (s)
n.d. n.d. ±
0.10 0.06 (s)
0.20 0.17 (s)
Pito FS DS t 0:01
3.6 2.8 3.1 1.2 (ns)d (ns)
0.31 0.26 (ns)
0.08 0.20 (s)
n.d. n.d. ±
0.11 0.06 (s)
0.13 0.08 (ns)
0.35 0.28 (s)
0.10 0.32 (s)
0.20 0.04 (s)
0.14 0.08 (s)
0.18 0.10 (s)
Burukutu FS 3.6 DS 3.2 t 0:01 (s) a b c d
3.1 1.1 (s)
n.d.=not detected; FS=fresh sample; DS=deteriorating sample. Means of triplicate determinations. s=signi®cant dierence between FS and DS (P40.01). ns=no signi®cant dierence between FS and DS (P>0.01).
groups of microorganisms. However, some of the isolates identi®ed, e.g. Flavobacterium and Alcaligenes are potentially pathogenic and could cause gastro-intestinal disorder. There was signi®cant dierence in the viable counts of microorganisms in the deteriorating beverage samples (Table 2). The reduction in the microbial population obtained for the deteriorating beverages indicated the gradual dying o of some initial micro-¯ora of the fermentation. There were signi®cant dierences in the lactic, malic, succinic and acetic acids content of the fresh and deteriorating sekete and burukutu. However, with the exception of malic and acetic, the results obtained for the pH, ethanol, lactic acid and succinic acid for pito samples were not signi®cantly dierent (Table 3). Decrease in the ethanol content of the deteriorating beverages is attributable to the activities of the Acetobacter spp. which oxidises the ethanol to acetic acid (Table 4). The proportional increase of the acetic acid of the deteriorating beer samples led credence to the above. It should be mentioned here that strict anaerobic
Table 4 Biochemical properties of Acetobacter species isolated from the deteriorating beverages (n=9)a Isolates Tests
A
B
C
D
E
F
G
H
I
J
Gram's reaction
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
ÿ
Cell dimension width (m) length (m) Morphology Catalase Nitrate reduction Acid from glucose Cellulose production Brown pigment Ethanol oxidation Growth in Hoyers medium
0.52 1.07 R + ÿ + ÿ ÿ + +
0.63 0.94 R + ÿ + ÿ ÿ + w+
0.71 1.23 R + ÿ + ÿ ÿ + +
0.56 0.86 O + ÿ ÿ ÿ ÿ + +
0.52 1.34 R + ÿ ÿ ÿ ÿ + +
0.71 1.21 R + ÿ + ÿ ÿ + +
0.62 0.94 R + ÿ + ÿ ÿ + ÿ
0.58 1.12 O + ÿ + ÿ ÿ + ÿ
0.52 0.81 R + ÿ + ÿ ÿ + ÿ
0.61 1.31 O + w+ ÿ ÿ ÿ + +
Ketogenesis from: Glycerol Mannitol Sorbitol
+ + +
+ ÿ +
+ + +
+ ÿ +
+ + +
+ + +
+ ÿ ÿ
ÿ + +
ÿ ÿ ÿ
ÿ ÿ ÿ
Ferric chloride reaction on: Glucose Fructose Galactose
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
ÿ ÿ ÿ
Growth on carbon sources: Ethanol Xylose Arabinose Glucose Ranose Sodium acetate Sodium citrate
+ ÿ + + ÿ + ÿ
+ + ÿ + ÿ + ÿ
+ ÿ ÿ + ÿ + ÿ
+ ÿ ÿ ÿ ÿ + ÿ
+ ÿ ÿ + ÿ + ÿ
+ ÿ ÿ + ÿ ÿ ÿ
+ ÿ ÿ + ÿ ÿ ÿ
+ ÿ ÿ + ÿ ÿ ÿ
+ + ÿ ÿ ÿ + ÿ
+ ÿ ÿ ÿ ÿ + ÿ
Growth on 0.001% malachite green Resistance to streptomycin
ÿ +
ÿ +
ÿ +
ÿ +
+ +
+ +
ÿ +
+ +
+ +
+ ÿ
Probability identity
AC
AC
AC
AC
AC
AH
AH
AH
AP
AP
a
R=rod; O=ovoid; ÿ=negative; +=positive; w+=weakly positive; AC=Acetobacter aceti; AH=Acetobacter hansenii; AP=Acetobacter pasteurianus.
A.I. Sanni et al. / Food Research International 32 (1999) 163±167
conditions are never achieved during the traditional brewing of these beverages, thus creating enabling environment for the acetic acid bacteria to become established and initiate spoilage. Despite the problem of spoilage, the production of these beverages oer an avenue for employment and income for the producers. According to Sefa-Dedeh and Asante (1988), production of pito in Ghana not only provides psychological and social enjoyment, but has important implications for the food system and economy of the country. Acknowledgements A.I.S. acknowledges the award of a Guest Scholarship by the Swedish Institute for research studies in the Department of Applied Microbiology, University of Lund, Sweden, where some aspects of this work were concluded. References Campbell-Platt, G. (1987). Fermented foods of the world, a dictionary guide. London: Butterworth, (pp. 193±195). Carr, J. G. (1968). Methods for identifying acetic acid bacteria. In B. M. Gibbs, & D.A Shapton, Identi®cation methods for microbiologists Part B. (p. 212). London, New York: Academic Press. Deak, T., & Beuchat, R. (1987). Identi®cation of food borne yeasts. Journal of Food Protection, 50, 243±264. Demuyakor, B, & Ohta, Y. (1991). Characteristics of pito yeast's from Ghana. Food Microbiology, 8, 183±193. Dey Ley, J., Gillis, M., & Swings, J. (1986). Family VI acetobacteraceae. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, & J. G. Holts, Bergey's manual of systematic bacteriology (pp. 267±278). Baltimore: Williams and Wilkins.
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